The present invention relates to methods for preparing factor X, activated factor X (factor Xa), inactivated factor X and inactivated factor Xa, compositions comprising factor X, factor Xa, inactivated factor X or inactivated factor Xa which are suitable for pharmaceutical use, and use of factor X, factor Xa, inactivated factor X or inactivated factor Xa for the treatment of various medical conditions.
Factor X is a coagulation factor normally present in human blood. Factor X deficiency is a rare bleeding disorder which affects between 1 in 500,000 and 1 in 1,000,000 of the population. It is characterised by a tendency to excessive bleeding, similar to that caused by factor VIII and factor IX deficiencies in haemophilia A and B respectively. There are currently no licensed treatments specifically for factor X deficiency anywhere in the world. In particular, there are no clinical concentrates of factor X currently available for use in the treatment of factor X deficiency. Instead, physicians have to rely on infusions of plasma or of prothrombin complex concentrates (PCC). However, there are a number of disadvantages accompanying use of plasma or PCC for the treatment of factor X deficiency.
Plasma contains only a low concentration of factor X (approximately 1 unit per mL), so a very large infusion volume is needed to achieve even a small increase in a patient's circulating level of factor X. Treatment is constrained by the need to stop infusion before a fully-therapeutic dose has been delivered, to avoid volume overload causing osmotic imbalance and transfusion-related acute lung injury. Plasma also requires refrigerated or frozen storage which can restrict availability to the patient.
Prothrombin complex concentrates (PCC) contain some factor X, but this is only a very minor component of the total protein present. PCCs are not assayed or labelled for this indication which leads to highly variable dosing. Many of these older products are made from pooled plasma without the added safety margin of multiple virus inactivation steps during manufacture. Most require refrigerated storage which, like plasma, can restrict availability to the patient. As the main constituent of PCC is prothrombin, and PCCs carry a risk of being partially activated by the manufacturing process, there is a risk that use of PCC will result in a thrombotic side effect during treatment, which could be fatal. Although PCC contain more factor X than plasma in the available volume, it is desirable to contain the therapeutic dose in as small a volume as possible, particularly as many of the treated patients are young children.
WO 89/05650 discloses a method for at least partially separating vitamin K-dependent blood clotting factors, including Factor X, from a mixture containing at least one such factor, for example a prothrombin complex concentrate. The method comprises adsorption of the mixture onto a metal chelate chromatography column. However, factor X produced according to the method in WO 89/05650 still contains significant amounts of prothrombin, up to 40-50% by weight. The presence of prothrombin in a concentrate of factor X is undesirable because prothrombin has a longer circulating half-life than factor X. If a patient is infused with treatment containing both proteins, this can cause a disproportionate and cumulative increase in plasma prothrombin which may then unbalance the haemostatic equilibrium in favour of thrombin generation and clot formation (haemostasis/thrombosis). Removal or reduction of prothrombin in a factor X concentrate would be desirable to minimise the risk of spontaneous thrombin generation during the manufacture or storage of the product, which could cause also thrombotic reactions during clinical use.
There is therefore still a need for a process for the preparation of factor X which is efficient, can be carried out on an industrial scale, which allows incorporation of multiple virus reduction steps and which provides a pharmaceutically useful product. There is also a need for pharmaceutical formulations of factor X for use in the treatment of conditions such as factor X deficiency.